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Mucosal infection and vaccination against feline immunodeficiency virus
Journal of Biotechnology 73 (1999) 213 – 221 www.elsevier.com/locate/jbiotec
Mucosal infection and vaccination against feline immunodeficiency virus C.R. Stokes *, S. Finerty, T.J. Gruffydd-Jones, C.P. Sturgess, D.A. Harbour Di6ision of Molecular and Cellular Biology, Uni6ersity of Bristol, Langford House, Langford, Bristol, North Somerset, BS40 5DU, UK Received 14 August 1998; received in revised form 4 January 1999; accepted 8 January 1999
Abstract Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8 + cells and greater levels of IL-2, IL-6, IL-10 and g-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Mucosal infection; Vaccination; Feline immunodeficiency virus * Corresponding author. Fax: + 44-117-9289505. E-mail address: [email protected] (C.R. Stokes) 0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 3 9 - X
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1. Introduction Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology, irrespective of the route of infection (Smith, 1994). Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes (Bishop et al., 1996).
2. Mucosal infection with FIV and lentiviral receptors The precise mechanisms of infection of lentiviruses via mucosal surfaces are unclear, but M cells (Amerongen et al., 1991), galactosyl ceramide on epithelial cells (Fantini et al., 1994), CD4 on lamina propria lymphocytes (LPLs) and Fc receptors on Langerhans cells (Hussain and Table 1 Mucosal vaccination schedulesa Antigens V3, linear peptide (RAISSWKQRNRWEWRPD) (200 mg) V3, multiple antigenic peptide (8 mer on lysine backbone) C2 Fixed virus (Gla-8, Pet), (2.5×107) Adju6ants Cholera toxin (10 mg) Quil A (20 mg) Iscoms Incomplete freunds (ICFA) Routes Intra-peritoneal (I.P.) Sub-cutaneous (S.C.) Rectal Intra-nasal Targeted lymph node (Internal iliac) a
Cats were vaccinated at weeks 0, 2 and 4. Cats were challenged rectally (ten MCID) at week 7 and monitored for virus isolation and PCR (proviral DNA and viral RNA).
Lehner, 1995) have all been implicated. The possible role of chemokine receptors in mucosal HIV infection has not been investigated, but it has been shown that rectal lamina propria cells express CCR5, and that epithelial cells can produce RANTES, MCP-1 and MIP-1b. The recent discovery that infection of cells with HIV depends on viral interaction with a specific co-receptor in addition to, or instead of, CD4 has promoted a re-evaluation of the value of FIV as a model for vaccine studies. Critics to the model had cited the evidence that FIV used CD9 as an alternative receptor as a major concern, but the recent data showing that FIV can also use CXCR4 in the entry process (Willett et al., 1997a) highlights that there may be greater similarities than was once thought. Indeed it would appear that the use of CXCR4 for entry may be a conserved feature of lentivirus biology (Willett et al., 1997b).
3. Mechanisms which control lentivirus infection Cytotoxic T lymphocytes (CTLs) in peripheral blood are known to be important in controlling HIV infection. In the acute infection the fall in the initial viremia coincides with the onset of the CTL response, which is then maintained until the terminal phase where progression to AIDS is seen (Koup et al., 1994). Also the detection of HIV-1 specific CTL responses in sero-negative ‘sex workers’ (Rowland-Jones et al., 1995) and babies born to infected mothers (Rowland-Jones et al., 1993) supports the notion that circulating CTLs might be responsible for the clearance of virus and protection from infection. In vaccinated cats, which are protected from challenge with FIV, Env-specific CTL have been detected in the peripheral blood (Flynn et al., 1996). In unvaccinated cats, peripheral blood CTL can also be transiently detected as early as 2 weeks post infection (Beatty et al., 1996), but in long term infected animals they become restricted to the lymph nodes and spleen (Flynn et al., 1996). The results of studies with vaccinated cats have indicated that virus-specific humoral and cellular immunity may play a role in protection. But that their relative importance may be dependent upon the interval
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Fig. 1. Serum IgG (open bars) and IgA (closed bars) antibody response to cholera toxin in groups of four cats immunized with V3 peptide and cholera toxin ( × 3) at 2-week intervals and assayed 3 weeks after final immunization. Cats were immunized via either sub-cutaneous, rectal or intra-nasal routes and the results expressed as percentage of standard serum. Control cats were unimmunized.
between vaccination and challenge (Hosie and Flynn, 1996). CD8 + T cells can also control HIV replication in CD4 + cells without killing the infected cell (Walker et al., 1986) by a non-MHC restricted mechanism. This non-cytotoxic anti-viral activity is mediated by a soluble factor (CAF) which lacks identity with any known cytokine (Levy et al., 1996). The non-cytotoxic CD8 + cells are detected in lymph nodes as well as blood and can suppress HIV replication at low CD8 + :CD4 + cell ratios. Whereas CD8 + CTL’s have been detected at all phases of viral infection non-cytotoxic CD8 + activity correlates directly with clinical state and the number of CD4 + cells in peripheral blood (Landay et al., 1993). Importantly similar observations have also been made in SIV- and HIV-infected non-human primates (Ennen et al., 1994).
4. Protection from lentiviral infection at mucosal surfaces Despite the large body of evidence to show the importance of the gastrointestinal tract in both the pathogenesis of HIV (Smith, 1994) and FIV infection (Bishop et al., 1996), relatively little is known of the immune mechanisms which control viral replication in mucosal tissue. In human patients infected with HIV it has been shown that whereas there is a significant decrease in the number of CD4 + T cells and RFD1 + dendritic cells, the number of CD8 + mucosal T cells is significantly increased in both the small (Lim et al., 1993a,b) and large intestine (Schneider et al., 1994). This increase was in both the CD45RO + and CD45RA + subsets but the former,
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CD8 + CD45RO + (memory) population remained dominant. Similarly in cats infected with FIV, we have shown there is an increase in the number of colonic lamina propria CD8 + cells (Sturgess, 1997). In order to begin to determine those mechanisms which may be important in controlling FIV replication within the gastrointestinal lamina propria, we have developed a procedure for the isolation of colonic LPLs and have studied asymptomatic cats which have been rectally infected with FIV for 1 year or longer. We could detect FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells (Bishop et al., 1996). Besides an increase in the number of lamina propria CD8 + cells we showed that infection resulted in an increase in the number of lamina propria
lymphocytes and greater levels of IL-2, IL-6, IL10 and g-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and CD8 + T cells may play a role in the control of local viral replication. Recent studies using the simian immunodeficiency virus (SIV)-macaque model have provided further evidence in support of a role for mucosal CD8 + cell populations in both the small intestine and rectal mucosa. In one study, small intestinal intraepithelial lymphocytes (IELs) from two chronically infected macaques showed greater direct SIV-specific cytotoxic activity (without in vitro re-stimulation), than cells obtained from other sites, including mesenteric lymph node. Interestingly, these macaques had been infected intravenously (Couedel-Courteille et al., 1997).
Table 2 Mucosal vaccination studiesa Antigen
Adjuvant
Routec
Outcome (% positive)b
1°
2° and 3°
CT CT Quil A Quil A – –
Rectal I.P. Rectal I.P. Rectal Rectal
Rectal Rectal Rectal Rectal Rectal Rectal
100 100 100 100 100 100
CT CT CT –
Rectal I.P. Intra-nasal Rectal
Rectal Rectal Rectal Rectal
100 100 100 100
IFA ISCOMS CT CT CT –
S.C. S.C. S.C. Rectal Intra-nasal S.C.
S.C. S.C. S.C. Rectal Intra-nasal S.C.
100 100 100 100 100 100
Control
Quil A Quil A –
TLN Rectal Rectal
TLN Rectal Rectal
100 100 100
Fixed FL4 Fixed FeTJ
Quil A Quil A
TLN TLN
TLN TLN
0 100
V3-MAP
Saline Fixed GLA-8
Control V3
Control C2
a Groups of cats (n\4) were immunised with FIV or FIV antigens as indicated in Table 1. Cats were challenged by rectal inoculation with purified virus or cell associated virus, and the outcome monitored by virus isolation on PBMCs. b The results of challenge up to 12 weeks are given as the cumulative percentage (%) virus isolation positive at weeks 3, 6 and 12. c I.P., Intra-peritoneal; S.C. sub-cutaneous.
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Anti-SIV CTL have also been detected in short term lines generated from lymphocytes isolated from rectal and cervico-vaginal mucosa from targeted lymph node immunised macaques (Klavinskis et al., 1996). In these animals protection was associated not only with the generation of CD8 + CTLs, but the production of CD8 + non-cytotoxic anti-viral factor (Klavinskis et al., 1996), IgA antibody-secreting cells, and the chemokines RANTES and MIP-1b. It would therefore appear that lentiviral infections can generate local CD8 responses (CTL and non-cytotoxic anti-viral factor) in both small and large intestine and that this is independent of the route of infection.
cell types present in the environment in which lamina propria T cells are triggered will affect the outcome of the response triggered. It is therefore not unreasonable to suppose that chronic lentivirus infection of intestinal cells may also profoundly influence mucosal responses. We have studied the cytokine profiles in LPLs isolated from asymptomatic cats infected rectally with FIV and shown that there were significantly higher levels of IL-2, IL-6 and IL-10 mRNA in ConA stimulated LPLs when compared to LPLs isolated from control cats (Sturgess, 1997).
5. Role of cytokines in disease progression
There is a large body of data to show that immune responses which are protective at mucosal surfaces are most effectively stimulated by local application of antigen. It is then no surprise that vaccination regimes which protect from parenteral lentiviral challenge generally fail to protect from rectal or vaginal challenge (Sutjipto et al., 1990). In contrast, recent attempts to vaccinate monkeys via the mucosal routes have produced encouraging results. For example, monkeys immunised with formalinised SIV in microparticles via a combination of intra-muscular and oral route showed a degree of protection from vaginal challenge (Marx et al., 1993), and animals repeatedly inoculated with a SHIV containing the envelope from HIV-1, were protected from vaginal challenge with pathogenic SIVmac239 (Miller et al., 1997). Using a recombinant SIV gp120 and p27 vaccine delivered via the targeted iliac lymph node route it has also been possible to protect from rectal challenge with the SIV molecular clone SIVmac 32HJ5 (Lehner et al., 1996). Mucosal vaccination is a rapidly developing area, and the recent promising results in both monkeys and cats indicate the potential of this approach for protection from mucosal infection with HIV and the reduction of viral load within this tissue. In order to exploit these advances we propose to compare a range of approaches for the delivery of viral antigens in stimulating both local humoral and cellular responses. Currently avail-
Cytokines play an important regulatory role in all immunological processes, and it has been suggested that a change in cytokine profile from a predominantly Th1 type to a Th2 type of response is seen in the progression of HIV infection towards AIDS (Clerici and Shearer, 1993). The profile of cytokines within the intestinal lamina propria would appear to be independent of other non-mucosal sites and to be highly sensitive to a range of factors. In the pig we have demonstrated that activation of isolated lamina propria T cells with concanavalin A (conA) resulted in a highly polarised response in which IL-4 was transcribed but not IL-2. The response of isolated human cells has been shown to be highly dependent upon the nature of the activation signal. For example it has been shown that ligation of the CD2 surface molecule resulted in secretion of much higher levels of IL-2, IL-4, gIFN and TNF-a by lamina propria cells than systemic cells (Targan et al., 1995), whilst co-ligation of CD3 plus CD28 triggered less IL-2 secretion. Other groups have reported that CD3/CD28 triggering resulted in higher levels of IL-4 secretion by intestinal than systemic cells (Boirivant et al., 1996), further indicating that lamina propria T cells have the potential to produce high levels of both IL-2 or IL-4, depending on the signal delivered during activation. Evidence from humans thus implies that the
6. Attempts to stimulate mucosal protective responses
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able data indicate that besides route of immunisation, there are a number of other factors that influence the chances for a mucosal vaccine to be successful. In particular it has been shown that live vaccines are more effective than non-replicating antigens that particles are more effective than soluble preparations, and that certain adjuvants are particularly effective. Several approaches have been developed which fulfil one or more of these criteria, including attenuated bacterial and viral vectors, biodegradable microparticles, ISCOMS, and a variety of mucosal adjuvants such as cholera toxin (Wilson et al., 1991) or muramyl dipeptide. Existing evidence suggests that the b subunit of V. cholerae toxin (CT-B) or interleukin-2 (IL-2) may be useful carrier molecules for antigenic moieties for mucosal vaccination since they also stimulate a good systemic response. For our own experiments on mucosal (rectal) vaccination of cats with FIV (Bishop et al., 1996) we have conjugated purified FIV virions to CT-B with the heterobifunctional reagent Sulpho-LC-SPDP. Vaccination by this method did not give sterilising protection from rectal challenge with FIV, but virus loads were significantly decreased (unpublished observations). A number of recent studies have shown that genetically produced chimaeric fusion proteins between antigens and mucosal adjuvants stimulate mucosal immunity. A particularly significant finding (Hazama et al., 1993) was that chimaeric fusion proteins between glycoprotein D (gD) of herpes simplex virus and either LT-B or IL-2 both stimulated mucosal and systemic immune responses when administered intra-nasally, but only the gD-IL-2 chimaera protected against systemic (intraperitoneal) challenge. Cytokines can influence the outcome of vaccination by exerting effects at various stages in the immune response, including enhancement of antigen presentation, polarisation of Th subsets, and regulation of antibody isotype production. For example it has been shown that whereas a mucosal IgA response may be enhanced by the use of IL-5 and IL-6 (Ramsay et al., 1994; Husband et al., 1996), IL-12 enhances vaccine induced immunity to Schistosoma mansoni by favouring the priming of a Th1 response (Montford et al.,
1996). Similarly, it has been shown that rIL-15 will augment the in vitro proliferation of CD3 + CD45 + cells from lepromatous patients to M. leprae (Jullien et al., 1997). The repeated finding that ‘adjuvant’ effects of cytokines could be observed when given either as a recombinant protein or as naked cytokine DNA (Pertmer et al., 1996), is of great significance, and the availability of sequence data for a number of feline cytokines will permit the application of this approach for the generation of mucosal responses to FIV. The use of naked DNA for vaccination has a number of advantages, not least of which is that the in vivo expression of proteins avoids the use of long and costly purification procedures. The endogenous expression of microbial proteins results in a presentation of antigens which favours the generation of cellular responses. In mice, injection of DNA coding for influenza virus nucleoprotein and haemagglutinin proteins stimulates not only a humoral response but also a strong CTL response leading to total protection (Ulmer et al., 1993). In macaques injection of DNA coding for SIV env and gag genes stimulates CTL responses. Recent studies in cats injected with DNA from a replication defective mutant of the FIV/F14 molecular clone with or without IFN-g DNA have produced encouraging results, with strong CTL activity and at least partial protection (Willett et al., 1997c). Similarly IL-12 DNA coadministered with HIV-1 antigens to mice resulted in a reduction in the specific antibody response, but a dramatic increase in specific CTLs (Kim et al., 1997). Comparison of immunisation with DNA given via either intra-muscular or epidermal route suggests that the outcome of the response differs (Pertmer et al., 1996), but to date there is no published data on the use of this approach, either with or without cytokine genes, to stimulate mucosal responses.
7. Mucosal vaccination studies with FIV We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with
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virulent virus. Cats have been immunised with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin, or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes (Table 1). Cats were immunised at weeks 0, 2 and 4 and challenged rectally at week 7 with ten CID of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (i.p. versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens (data not shown). However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route (Fig. 1). All cats immunised via intraperitonal, sub-cutaneous, rectal or intra-nasal routes became virus isolation positive following rectal challenge with FIV. Cats immunised with the FIV C2 peptide via the targeted lymph node route (internal ileac lymph node) also became virus isolation positive. In contrast four cats immunised (×3) with FL4 cells (a FIV infected cell line) by the targeted lymph node route were protected. Control cats similarly immunised with FetJ cells (uninfected cells from the same origin as FL4) all became virus isolation positive (Table 2). Analysis of the humoral and cellular (proliferative and CTL responses) responses would indicate that targeted lymph node (TLN) was more effective that rectal immunisation in stimulating both local and systemic responses. Currently experiments are in progress to confirm these preliminary observations and to determine the local protective mechanisms.
Acknowledgements This work was supported by grants from the Medical Research Council of the United Kingdom.
References Amerongen, H.M., Weltzin, R., Farnet, C.M., Michetti, P., Haseltine, W.A., Neytra, M.R., 1991. Transepithelial
219
transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS. J. Acquir. Immun. Defic. Synd. 4, 760 – 765. Beatty, J.A., Willett, B.J., Gault, E.A., Jarrett, O., 1996. A longitudinal study of feline immunodeficiency virus-specific cytotoxic T lymphocytes in experimentally infected cats, using antigen-specific induction. J. Virol. 70, 6199 – 6206. Bishop, S.A., Stokes, C.R., Gruffydd-Jones, T.J., Whiting, C.V., Harbour, D.A., 1996. Vaginal and rectal infection of cats with feline immunodeficiency virus. Vet. Microbiol. 51, 217 – 227. Boirivant, M., Fuss, I., Fiocchi, C., Kleins, S., Strong, S.A., Strober, W., 1996. Hypoproliferative human lamina propria T-cells retain their capacity to secrete lymphokines when stimulated via CD2/CD28 pathways. Proc. Assoc. Am. Physicians 108, 55 – 67. Clerici, M., Shearer, G.M., 1993. A TH1 TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14, 107 – 111. Couedel-Courteille, A., Le Grand, R., Tulliez, M., Guillet, J.-G., Venet, A., 1997. Direct ex vivo simian immunodeficiency virus (SIV)-specific cytotoxic activity detected from small intestine intraepithelial lymphocytes of SIV-infected macaques at an advanced stage of infection. J. Virol. 71, 1052 – 1057. Ennen, J., Findeklee, H., Dittmar, M.T., Norley, S., Ernst, M., Kurth, R., 1994. CD8 + T-lymphocytes of African green monkeys secrete an immunodeficiency virus suppressing factors. Proc. Natl. Acad. Sci. 91, 7207 – 7211. Fantini, J., Yahi, N., Delezay, O., Gonzalez-Scarano, F., 1994. GalCer, CD26 and HIV infection of intestinal epithelial cells. AIDS 8, 1347 – 1348. Flynn, J.N., Keating, P., Hosie, M.J., Macket, M., Stephen, E.B., Beatty, J.A., Neil, J.C., Jarrett, O., 1996. Env-specific CTL predominate in cats protected from feline immunodeficiency virus infection by vaccination. J. Immunol. 157, 3658 – 3665. Hazama, M., Mayumi-Aono, A., Miyazaki, T., Hinuma, S., Fujisawa, Y., 1993. Intranasal immunization against herpes simplex virus infection by using a recombinant glycoprotein D fused with immunomodulating proteins, the b subunit of Escherichia coli heat labile enterotoxin and interleukin. Immunology 78, 643 – 649. Hosie, M.J., Flynn, J.N., 1996. Feline immunodeficiency virus vaccination: characterisation of the immune correlation of protection. J. Virol. 70, 7561 – 7568. Husband, A.J., Kramer, D.R., Bao, S., Sutherland, R.M., Beagley, K.W., 1996. Regulation of mucosal IgA responses in vivo: cytokines and adjuvants. Vet. Immunol. Immunopathol. 54, 179 – 186. Hussain, L.A., Lehner, T., 1995. Comparative investigation of Langerhans cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. Immunology 85, 475 – 484. Jullien, D., Sieling, P.A., Uyemura, K., Mar, N.D., Rea, T.H., Modlin, R.L., 1997. IL-15 an immunomodulator of T cell responses in intracellular infection. J. Immunol. 158, 800 – 806.
220
C.R. Stokes et al. / Journal of Biotechnology 73 (1999) 213–221
Kim, J.J., Ayyavoo, V., Bagarazzi, M.L., Chattergoon, M.A., Dang, K., Wang, B., Boyer, J.D., Weiner, D.B., 1997. In vivo engineering of a cellular immune response by co-administration of IL-12 expression vector with a DNA immunogen. J. Immunol. 158, 816–826. Klavinskis, L.S., Bergmeier, L.A., Gao, L., Mitchell, E., Ward, R.G., Layton, G., Brookes, R., Meyers, N.J., Lehner, T., 1996. Mucosal or targeted lymph node immunization of macaques with a particulate SIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes. J. Immunol. 157, 2521– 2527. Koup, R.A., Safrit, J.T., Cao, Y., Andrews, Ca., Mcleod, G., Borkowsky, W., Farthing, C., Ho, D.D., 1994. Temporal associates of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type-1 syndrome. J. Virol. 68, 4650–4655. Landay, A.L., Mackewicz, C., Levy, J.A., 1993. An activated CD8 + T-cell phenotype correlates with anti-HIV activity and asymptomatic clinical states. Clin. Immunol. Immunopathol. 69, 106–116. Lehner, T., Wang, Y.F., Cranage, M., Bergmeier, L.A., Mitchell, E., Hall, G., Dennis, M., Cook, N., Brookes, R., Klavinkis, L., Doyle, C., Ward, R., 1996. Protective mucosal immunity elicited by targeted iliac lymph node immunisation with a subunit SIV envelope and core vaccines in macaques. Nat. Med. 2, 767–775. Levy, J.A., Mackewicz, C.E., Barker, E., 1996. Controlling HIV pathogenesis: the role of the non-cytotoxic anti-HIV response of CD8 + T-cells. Immunol. Today 17, 217– 224. Lim, S.G., Condez, A., Lee, C.A., Johnson, M.A., Elia, C., Poulter, L.W., 1993a. Loss of mucosal CD4 lymphocytes is an early feature of HIV infection. Clin. Exp. Immunol. 92, 448 – 454. Lim, S.G., Condez, A., Poulter, L.W., 1993b. Mucosal macrophage subsets of the gut in HIV: decrease in antigen presenting cell phenotype. Clin. Exp. Immunol. 92, 442 – 447. Marx, P.A., Compans, R.W., Gettie, A., Staas, J.K., Gilley, R.M., Mulligan, M.J., Yamschikov, G.V., Chen, D., Eldridge, J.H., 1993. Protection against vaginal SIV transmission with encapsulated vaccine. Science 260, 1323 – 1327. Miller, C.J., McChesney, M.B., Lu, X., Dailey, P.J., Chutkowski, C., Brosio, P., Roberts, B., Lu, Y., 1997. Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from pathogenic vaginal challenge with pathogenic SIVmac239. J. Virol. 71, 1191 – 1921. Montford, A.P., Anderson, S., Wilson, R.A., 1996. Induction of Th1 cell mediated protective immunity to Schistosoma mansoni by co-administration of larval antigens and IL-12 as an adjuvant. J. Immunol. 156, 4739–4745. Pertmer, T.M., Roberts, T.R., Haynes, J.R., 1996. Influenza
virus nucleoprotein specific immunoglobulin-G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. J. Virol. 70, 6119 – 6125. Ramsay, A.J., Leong, K.H., Boyle, D., Ruby, J., Ramshaw, I.A., 1994. Enhancement of mucosal IgA responses by interleukin-5 and interleukin-6 encoded in recombinant vaccine vectors. Reprod. Fertil. Dev. 6, 389 – 392. Rowland-Jones, S.L., Nixon, D.F., Aldhous, M.C., Gotch, F., Ariyoshi, K., Hallam, N., Kroll, J.S., Froebel, K., McMichael, A., 1993. HIV-specific T-cell activity in an HIV-exposed but uninfected infant. Lancet 341, 860 – 861. Rowland-Jones, S.L., Sutton, J., Ariyoshi, K., Dong, T., Gotch, F., Mcadam, S., Whitby, D., Sabally, S., Gallimore, A., Corrah, T., Takiguchi, M., Schultz, T., McMichael, A., Whittle, H., 1995. HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women. Nat. Med. 1, 59 – 64. Schneider, T., Ullrich, K., Bergs, C., Schmidt, W., Riecken, E.O., Zeitz, M., 1994. Abnormalities in subset distribution, activation and differentiation of T-cells isolated from laarge intestine biopsies in HIV infection. Clin. Exp. Immunol. 95, 430 – 435. Smith, P.D., 1994. Role of the mucosa in human immunodeficiency virus disease. Mucosal Immunol. Update 2, 3 – 6. Sturgess, C.P., 1997. Studies on mucosal effector mechanisms in feline immunodeficiency virus (FIV) infection in cats. PhD thesis, University of Bristol. Sutjipto, S., Pedersen, N.C., Miller, C.J., Gardner, M.B., Hanson, C.V., Gettie, A., Jennings, M., Higgins, J., Marx, P.A., 1990. Inactivated simian immunodeficiency virus-vaccine failed to protect rhesus macaques from intravenous or genital mucosal infection but delayed disease in intravenously exposed animals. J. Virol. 64, 2290 – 2297. Targan, S.R., Deem, R.L., Liu, M., Wang, S., Nel, A., 1995. Definition of a lamina propria T-cell responsive state: enhanced cytokine responsiveness of T-cells stimulated through CD2 pathway. J. Immunol. 154, 664 – 675. Ulmer, J.B., Donnelly, J.J., Parker, S.E., Rhodes, G.H., Felgner, P.L., Dwarki, V.J., Gromkowski, S.H., Deck, R.R., Dewitt, C.M., Friedman, A, Hawe, L.A., Leander, K.R., Martinez, D., Perry, H.C., Shiver, J.W., Montgomery, D.L., Liu, M.A., 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259, 1745 – 1749. Walker, C.M., Moody, D.J., Stites, D.P., Levy, J.A., 1986. Lymphocytes-CD8 + can control HIV infection in vitro by suppressing virus replication. Science 234, 1563 – 1566. Willett, B.J., Picard, L., Hosie, M.J., Turner, J.D., Adema, K., Clapham, P.R., 1997a. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. J. Virol. 71, 6407 – 6415. Willett, B.J., Hosie, M.J., Neil, J.C., Turner, J.D., Hoxie,
C.R. Stokes et al. / Journal of Biotechnology 73 (1999) 213–221 J.A., 1997b. Common mechanisms of infection by lentiviruses. Nature 385, 587. Willett, B.J., Flynn, J.N., Hosie, M.J., 1997c. FIV infection of the domestic cat: an animal model for AIDS. Immunol. Today 18, 182 – 188.
.
221
Wilson, A.D., Bailey, M., Williams, N.A., Stokes, C.R., 1991. The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocvanin using cholera toxin as an adjuvant. Eur. J. Immunol. 21, 2333 – 2339.
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Mucosal infection and vaccination against feline immunodeficiency virus C.R. Stokes *, S. Finerty, T.J. Gruffydd-Jones, C.P. Sturgess, D.A. Harbour Di6ision of Molecular and Cellular Biology, Uni6ersity of Bristol, Langford House, Langford, Bristol, North Somerset, BS40 5DU, UK Received 14 August 1998; received in revised form 4 January 1999; accepted 8 January 1999
Abstract Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8 + cells and greater levels of IL-2, IL-6, IL-10 and g-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Mucosal infection; Vaccination; Feline immunodeficiency virus * Corresponding author. Fax: + 44-117-9289505. E-mail address: [email protected] (C.R. Stokes) 0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 3 9 - X
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1. Introduction Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology, irrespective of the route of infection (Smith, 1994). Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes (Bishop et al., 1996).
2. Mucosal infection with FIV and lentiviral receptors The precise mechanisms of infection of lentiviruses via mucosal surfaces are unclear, but M cells (Amerongen et al., 1991), galactosyl ceramide on epithelial cells (Fantini et al., 1994), CD4 on lamina propria lymphocytes (LPLs) and Fc receptors on Langerhans cells (Hussain and Table 1 Mucosal vaccination schedulesa Antigens V3, linear peptide (RAISSWKQRNRWEWRPD) (200 mg) V3, multiple antigenic peptide (8 mer on lysine backbone) C2 Fixed virus (Gla-8, Pet), (2.5×107) Adju6ants Cholera toxin (10 mg) Quil A (20 mg) Iscoms Incomplete freunds (ICFA) Routes Intra-peritoneal (I.P.) Sub-cutaneous (S.C.) Rectal Intra-nasal Targeted lymph node (Internal iliac) a
Cats were vaccinated at weeks 0, 2 and 4. Cats were challenged rectally (ten MCID) at week 7 and monitored for virus isolation and PCR (proviral DNA and viral RNA).
Lehner, 1995) have all been implicated. The possible role of chemokine receptors in mucosal HIV infection has not been investigated, but it has been shown that rectal lamina propria cells express CCR5, and that epithelial cells can produce RANTES, MCP-1 and MIP-1b. The recent discovery that infection of cells with HIV depends on viral interaction with a specific co-receptor in addition to, or instead of, CD4 has promoted a re-evaluation of the value of FIV as a model for vaccine studies. Critics to the model had cited the evidence that FIV used CD9 as an alternative receptor as a major concern, but the recent data showing that FIV can also use CXCR4 in the entry process (Willett et al., 1997a) highlights that there may be greater similarities than was once thought. Indeed it would appear that the use of CXCR4 for entry may be a conserved feature of lentivirus biology (Willett et al., 1997b).
3. Mechanisms which control lentivirus infection Cytotoxic T lymphocytes (CTLs) in peripheral blood are known to be important in controlling HIV infection. In the acute infection the fall in the initial viremia coincides with the onset of the CTL response, which is then maintained until the terminal phase where progression to AIDS is seen (Koup et al., 1994). Also the detection of HIV-1 specific CTL responses in sero-negative ‘sex workers’ (Rowland-Jones et al., 1995) and babies born to infected mothers (Rowland-Jones et al., 1993) supports the notion that circulating CTLs might be responsible for the clearance of virus and protection from infection. In vaccinated cats, which are protected from challenge with FIV, Env-specific CTL have been detected in the peripheral blood (Flynn et al., 1996). In unvaccinated cats, peripheral blood CTL can also be transiently detected as early as 2 weeks post infection (Beatty et al., 1996), but in long term infected animals they become restricted to the lymph nodes and spleen (Flynn et al., 1996). The results of studies with vaccinated cats have indicated that virus-specific humoral and cellular immunity may play a role in protection. But that their relative importance may be dependent upon the interval
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Fig. 1. Serum IgG (open bars) and IgA (closed bars) antibody response to cholera toxin in groups of four cats immunized with V3 peptide and cholera toxin ( × 3) at 2-week intervals and assayed 3 weeks after final immunization. Cats were immunized via either sub-cutaneous, rectal or intra-nasal routes and the results expressed as percentage of standard serum. Control cats were unimmunized.
between vaccination and challenge (Hosie and Flynn, 1996). CD8 + T cells can also control HIV replication in CD4 + cells without killing the infected cell (Walker et al., 1986) by a non-MHC restricted mechanism. This non-cytotoxic anti-viral activity is mediated by a soluble factor (CAF) which lacks identity with any known cytokine (Levy et al., 1996). The non-cytotoxic CD8 + cells are detected in lymph nodes as well as blood and can suppress HIV replication at low CD8 + :CD4 + cell ratios. Whereas CD8 + CTL’s have been detected at all phases of viral infection non-cytotoxic CD8 + activity correlates directly with clinical state and the number of CD4 + cells in peripheral blood (Landay et al., 1993). Importantly similar observations have also been made in SIV- and HIV-infected non-human primates (Ennen et al., 1994).
4. Protection from lentiviral infection at mucosal surfaces Despite the large body of evidence to show the importance of the gastrointestinal tract in both the pathogenesis of HIV (Smith, 1994) and FIV infection (Bishop et al., 1996), relatively little is known of the immune mechanisms which control viral replication in mucosal tissue. In human patients infected with HIV it has been shown that whereas there is a significant decrease in the number of CD4 + T cells and RFD1 + dendritic cells, the number of CD8 + mucosal T cells is significantly increased in both the small (Lim et al., 1993a,b) and large intestine (Schneider et al., 1994). This increase was in both the CD45RO + and CD45RA + subsets but the former,
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CD8 + CD45RO + (memory) population remained dominant. Similarly in cats infected with FIV, we have shown there is an increase in the number of colonic lamina propria CD8 + cells (Sturgess, 1997). In order to begin to determine those mechanisms which may be important in controlling FIV replication within the gastrointestinal lamina propria, we have developed a procedure for the isolation of colonic LPLs and have studied asymptomatic cats which have been rectally infected with FIV for 1 year or longer. We could detect FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells (Bishop et al., 1996). Besides an increase in the number of lamina propria CD8 + cells we showed that infection resulted in an increase in the number of lamina propria
lymphocytes and greater levels of IL-2, IL-6, IL10 and g-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and CD8 + T cells may play a role in the control of local viral replication. Recent studies using the simian immunodeficiency virus (SIV)-macaque model have provided further evidence in support of a role for mucosal CD8 + cell populations in both the small intestine and rectal mucosa. In one study, small intestinal intraepithelial lymphocytes (IELs) from two chronically infected macaques showed greater direct SIV-specific cytotoxic activity (without in vitro re-stimulation), than cells obtained from other sites, including mesenteric lymph node. Interestingly, these macaques had been infected intravenously (Couedel-Courteille et al., 1997).
Table 2 Mucosal vaccination studiesa Antigen
Adjuvant
Routec
Outcome (% positive)b
1°
2° and 3°
CT CT Quil A Quil A – –
Rectal I.P. Rectal I.P. Rectal Rectal
Rectal Rectal Rectal Rectal Rectal Rectal
100 100 100 100 100 100
CT CT CT –
Rectal I.P. Intra-nasal Rectal
Rectal Rectal Rectal Rectal
100 100 100 100
IFA ISCOMS CT CT CT –
S.C. S.C. S.C. Rectal Intra-nasal S.C.
S.C. S.C. S.C. Rectal Intra-nasal S.C.
100 100 100 100 100 100
Control
Quil A Quil A –
TLN Rectal Rectal
TLN Rectal Rectal
100 100 100
Fixed FL4 Fixed FeTJ
Quil A Quil A
TLN TLN
TLN TLN
0 100
V3-MAP
Saline Fixed GLA-8
Control V3
Control C2
a Groups of cats (n\4) were immunised with FIV or FIV antigens as indicated in Table 1. Cats were challenged by rectal inoculation with purified virus or cell associated virus, and the outcome monitored by virus isolation on PBMCs. b The results of challenge up to 12 weeks are given as the cumulative percentage (%) virus isolation positive at weeks 3, 6 and 12. c I.P., Intra-peritoneal; S.C. sub-cutaneous.
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Anti-SIV CTL have also been detected in short term lines generated from lymphocytes isolated from rectal and cervico-vaginal mucosa from targeted lymph node immunised macaques (Klavinskis et al., 1996). In these animals protection was associated not only with the generation of CD8 + CTLs, but the production of CD8 + non-cytotoxic anti-viral factor (Klavinskis et al., 1996), IgA antibody-secreting cells, and the chemokines RANTES and MIP-1b. It would therefore appear that lentiviral infections can generate local CD8 responses (CTL and non-cytotoxic anti-viral factor) in both small and large intestine and that this is independent of the route of infection.
cell types present in the environment in which lamina propria T cells are triggered will affect the outcome of the response triggered. It is therefore not unreasonable to suppose that chronic lentivirus infection of intestinal cells may also profoundly influence mucosal responses. We have studied the cytokine profiles in LPLs isolated from asymptomatic cats infected rectally with FIV and shown that there were significantly higher levels of IL-2, IL-6 and IL-10 mRNA in ConA stimulated LPLs when compared to LPLs isolated from control cats (Sturgess, 1997).
5. Role of cytokines in disease progression
There is a large body of data to show that immune responses which are protective at mucosal surfaces are most effectively stimulated by local application of antigen. It is then no surprise that vaccination regimes which protect from parenteral lentiviral challenge generally fail to protect from rectal or vaginal challenge (Sutjipto et al., 1990). In contrast, recent attempts to vaccinate monkeys via the mucosal routes have produced encouraging results. For example, monkeys immunised with formalinised SIV in microparticles via a combination of intra-muscular and oral route showed a degree of protection from vaginal challenge (Marx et al., 1993), and animals repeatedly inoculated with a SHIV containing the envelope from HIV-1, were protected from vaginal challenge with pathogenic SIVmac239 (Miller et al., 1997). Using a recombinant SIV gp120 and p27 vaccine delivered via the targeted iliac lymph node route it has also been possible to protect from rectal challenge with the SIV molecular clone SIVmac 32HJ5 (Lehner et al., 1996). Mucosal vaccination is a rapidly developing area, and the recent promising results in both monkeys and cats indicate the potential of this approach for protection from mucosal infection with HIV and the reduction of viral load within this tissue. In order to exploit these advances we propose to compare a range of approaches for the delivery of viral antigens in stimulating both local humoral and cellular responses. Currently avail-
Cytokines play an important regulatory role in all immunological processes, and it has been suggested that a change in cytokine profile from a predominantly Th1 type to a Th2 type of response is seen in the progression of HIV infection towards AIDS (Clerici and Shearer, 1993). The profile of cytokines within the intestinal lamina propria would appear to be independent of other non-mucosal sites and to be highly sensitive to a range of factors. In the pig we have demonstrated that activation of isolated lamina propria T cells with concanavalin A (conA) resulted in a highly polarised response in which IL-4 was transcribed but not IL-2. The response of isolated human cells has been shown to be highly dependent upon the nature of the activation signal. For example it has been shown that ligation of the CD2 surface molecule resulted in secretion of much higher levels of IL-2, IL-4, gIFN and TNF-a by lamina propria cells than systemic cells (Targan et al., 1995), whilst co-ligation of CD3 plus CD28 triggered less IL-2 secretion. Other groups have reported that CD3/CD28 triggering resulted in higher levels of IL-4 secretion by intestinal than systemic cells (Boirivant et al., 1996), further indicating that lamina propria T cells have the potential to produce high levels of both IL-2 or IL-4, depending on the signal delivered during activation. Evidence from humans thus implies that the
6. Attempts to stimulate mucosal protective responses
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able data indicate that besides route of immunisation, there are a number of other factors that influence the chances for a mucosal vaccine to be successful. In particular it has been shown that live vaccines are more effective than non-replicating antigens that particles are more effective than soluble preparations, and that certain adjuvants are particularly effective. Several approaches have been developed which fulfil one or more of these criteria, including attenuated bacterial and viral vectors, biodegradable microparticles, ISCOMS, and a variety of mucosal adjuvants such as cholera toxin (Wilson et al., 1991) or muramyl dipeptide. Existing evidence suggests that the b subunit of V. cholerae toxin (CT-B) or interleukin-2 (IL-2) may be useful carrier molecules for antigenic moieties for mucosal vaccination since they also stimulate a good systemic response. For our own experiments on mucosal (rectal) vaccination of cats with FIV (Bishop et al., 1996) we have conjugated purified FIV virions to CT-B with the heterobifunctional reagent Sulpho-LC-SPDP. Vaccination by this method did not give sterilising protection from rectal challenge with FIV, but virus loads were significantly decreased (unpublished observations). A number of recent studies have shown that genetically produced chimaeric fusion proteins between antigens and mucosal adjuvants stimulate mucosal immunity. A particularly significant finding (Hazama et al., 1993) was that chimaeric fusion proteins between glycoprotein D (gD) of herpes simplex virus and either LT-B or IL-2 both stimulated mucosal and systemic immune responses when administered intra-nasally, but only the gD-IL-2 chimaera protected against systemic (intraperitoneal) challenge. Cytokines can influence the outcome of vaccination by exerting effects at various stages in the immune response, including enhancement of antigen presentation, polarisation of Th subsets, and regulation of antibody isotype production. For example it has been shown that whereas a mucosal IgA response may be enhanced by the use of IL-5 and IL-6 (Ramsay et al., 1994; Husband et al., 1996), IL-12 enhances vaccine induced immunity to Schistosoma mansoni by favouring the priming of a Th1 response (Montford et al.,
1996). Similarly, it has been shown that rIL-15 will augment the in vitro proliferation of CD3 + CD45 + cells from lepromatous patients to M. leprae (Jullien et al., 1997). The repeated finding that ‘adjuvant’ effects of cytokines could be observed when given either as a recombinant protein or as naked cytokine DNA (Pertmer et al., 1996), is of great significance, and the availability of sequence data for a number of feline cytokines will permit the application of this approach for the generation of mucosal responses to FIV. The use of naked DNA for vaccination has a number of advantages, not least of which is that the in vivo expression of proteins avoids the use of long and costly purification procedures. The endogenous expression of microbial proteins results in a presentation of antigens which favours the generation of cellular responses. In mice, injection of DNA coding for influenza virus nucleoprotein and haemagglutinin proteins stimulates not only a humoral response but also a strong CTL response leading to total protection (Ulmer et al., 1993). In macaques injection of DNA coding for SIV env and gag genes stimulates CTL responses. Recent studies in cats injected with DNA from a replication defective mutant of the FIV/F14 molecular clone with or without IFN-g DNA have produced encouraging results, with strong CTL activity and at least partial protection (Willett et al., 1997c). Similarly IL-12 DNA coadministered with HIV-1 antigens to mice resulted in a reduction in the specific antibody response, but a dramatic increase in specific CTLs (Kim et al., 1997). Comparison of immunisation with DNA given via either intra-muscular or epidermal route suggests that the outcome of the response differs (Pertmer et al., 1996), but to date there is no published data on the use of this approach, either with or without cytokine genes, to stimulate mucosal responses.
7. Mucosal vaccination studies with FIV We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with
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virulent virus. Cats have been immunised with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin, or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes (Table 1). Cats were immunised at weeks 0, 2 and 4 and challenged rectally at week 7 with ten CID of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (i.p. versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens (data not shown). However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route (Fig. 1). All cats immunised via intraperitonal, sub-cutaneous, rectal or intra-nasal routes became virus isolation positive following rectal challenge with FIV. Cats immunised with the FIV C2 peptide via the targeted lymph node route (internal ileac lymph node) also became virus isolation positive. In contrast four cats immunised (×3) with FL4 cells (a FIV infected cell line) by the targeted lymph node route were protected. Control cats similarly immunised with FetJ cells (uninfected cells from the same origin as FL4) all became virus isolation positive (Table 2). Analysis of the humoral and cellular (proliferative and CTL responses) responses would indicate that targeted lymph node (TLN) was more effective that rectal immunisation in stimulating both local and systemic responses. Currently experiments are in progress to confirm these preliminary observations and to determine the local protective mechanisms.
Acknowledgements This work was supported by grants from the Medical Research Council of the United Kingdom.
References Amerongen, H.M., Weltzin, R., Farnet, C.M., Michetti, P., Haseltine, W.A., Neytra, M.R., 1991. Transepithelial
219
transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS. J. Acquir. Immun. Defic. Synd. 4, 760 – 765. Beatty, J.A., Willett, B.J., Gault, E.A., Jarrett, O., 1996. A longitudinal study of feline immunodeficiency virus-specific cytotoxic T lymphocytes in experimentally infected cats, using antigen-specific induction. J. Virol. 70, 6199 – 6206. Bishop, S.A., Stokes, C.R., Gruffydd-Jones, T.J., Whiting, C.V., Harbour, D.A., 1996. Vaginal and rectal infection of cats with feline immunodeficiency virus. Vet. Microbiol. 51, 217 – 227. Boirivant, M., Fuss, I., Fiocchi, C., Kleins, S., Strong, S.A., Strober, W., 1996. Hypoproliferative human lamina propria T-cells retain their capacity to secrete lymphokines when stimulated via CD2/CD28 pathways. Proc. Assoc. Am. Physicians 108, 55 – 67. Clerici, M., Shearer, G.M., 1993. A TH1 TH2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14, 107 – 111. Couedel-Courteille, A., Le Grand, R., Tulliez, M., Guillet, J.-G., Venet, A., 1997. Direct ex vivo simian immunodeficiency virus (SIV)-specific cytotoxic activity detected from small intestine intraepithelial lymphocytes of SIV-infected macaques at an advanced stage of infection. J. Virol. 71, 1052 – 1057. Ennen, J., Findeklee, H., Dittmar, M.T., Norley, S., Ernst, M., Kurth, R., 1994. CD8 + T-lymphocytes of African green monkeys secrete an immunodeficiency virus suppressing factors. Proc. Natl. Acad. Sci. 91, 7207 – 7211. Fantini, J., Yahi, N., Delezay, O., Gonzalez-Scarano, F., 1994. GalCer, CD26 and HIV infection of intestinal epithelial cells. AIDS 8, 1347 – 1348. Flynn, J.N., Keating, P., Hosie, M.J., Macket, M., Stephen, E.B., Beatty, J.A., Neil, J.C., Jarrett, O., 1996. Env-specific CTL predominate in cats protected from feline immunodeficiency virus infection by vaccination. J. Immunol. 157, 3658 – 3665. Hazama, M., Mayumi-Aono, A., Miyazaki, T., Hinuma, S., Fujisawa, Y., 1993. Intranasal immunization against herpes simplex virus infection by using a recombinant glycoprotein D fused with immunomodulating proteins, the b subunit of Escherichia coli heat labile enterotoxin and interleukin. Immunology 78, 643 – 649. Hosie, M.J., Flynn, J.N., 1996. Feline immunodeficiency virus vaccination: characterisation of the immune correlation of protection. J. Virol. 70, 7561 – 7568. Husband, A.J., Kramer, D.R., Bao, S., Sutherland, R.M., Beagley, K.W., 1996. Regulation of mucosal IgA responses in vivo: cytokines and adjuvants. Vet. Immunol. Immunopathol. 54, 179 – 186. Hussain, L.A., Lehner, T., 1995. Comparative investigation of Langerhans cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. Immunology 85, 475 – 484. Jullien, D., Sieling, P.A., Uyemura, K., Mar, N.D., Rea, T.H., Modlin, R.L., 1997. IL-15 an immunomodulator of T cell responses in intracellular infection. J. Immunol. 158, 800 – 806.
220
C.R. Stokes et al. / Journal of Biotechnology 73 (1999) 213–221
Kim, J.J., Ayyavoo, V., Bagarazzi, M.L., Chattergoon, M.A., Dang, K., Wang, B., Boyer, J.D., Weiner, D.B., 1997. In vivo engineering of a cellular immune response by co-administration of IL-12 expression vector with a DNA immunogen. J. Immunol. 158, 816–826. Klavinskis, L.S., Bergmeier, L.A., Gao, L., Mitchell, E., Ward, R.G., Layton, G., Brookes, R., Meyers, N.J., Lehner, T., 1996. Mucosal or targeted lymph node immunization of macaques with a particulate SIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes. J. Immunol. 157, 2521– 2527. Koup, R.A., Safrit, J.T., Cao, Y., Andrews, Ca., Mcleod, G., Borkowsky, W., Farthing, C., Ho, D.D., 1994. Temporal associates of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type-1 syndrome. J. Virol. 68, 4650–4655. Landay, A.L., Mackewicz, C., Levy, J.A., 1993. An activated CD8 + T-cell phenotype correlates with anti-HIV activity and asymptomatic clinical states. Clin. Immunol. Immunopathol. 69, 106–116. Lehner, T., Wang, Y.F., Cranage, M., Bergmeier, L.A., Mitchell, E., Hall, G., Dennis, M., Cook, N., Brookes, R., Klavinkis, L., Doyle, C., Ward, R., 1996. Protective mucosal immunity elicited by targeted iliac lymph node immunisation with a subunit SIV envelope and core vaccines in macaques. Nat. Med. 2, 767–775. Levy, J.A., Mackewicz, C.E., Barker, E., 1996. Controlling HIV pathogenesis: the role of the non-cytotoxic anti-HIV response of CD8 + T-cells. Immunol. Today 17, 217– 224. Lim, S.G., Condez, A., Lee, C.A., Johnson, M.A., Elia, C., Poulter, L.W., 1993a. Loss of mucosal CD4 lymphocytes is an early feature of HIV infection. Clin. Exp. Immunol. 92, 448 – 454. Lim, S.G., Condez, A., Poulter, L.W., 1993b. Mucosal macrophage subsets of the gut in HIV: decrease in antigen presenting cell phenotype. Clin. Exp. Immunol. 92, 442 – 447. Marx, P.A., Compans, R.W., Gettie, A., Staas, J.K., Gilley, R.M., Mulligan, M.J., Yamschikov, G.V., Chen, D., Eldridge, J.H., 1993. Protection against vaginal SIV transmission with encapsulated vaccine. Science 260, 1323 – 1327. Miller, C.J., McChesney, M.B., Lu, X., Dailey, P.J., Chutkowski, C., Brosio, P., Roberts, B., Lu, Y., 1997. Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from pathogenic vaginal challenge with pathogenic SIVmac239. J. Virol. 71, 1191 – 1921. Montford, A.P., Anderson, S., Wilson, R.A., 1996. Induction of Th1 cell mediated protective immunity to Schistosoma mansoni by co-administration of larval antigens and IL-12 as an adjuvant. J. Immunol. 156, 4739–4745. Pertmer, T.M., Roberts, T.R., Haynes, J.R., 1996. Influenza
virus nucleoprotein specific immunoglobulin-G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. J. Virol. 70, 6119 – 6125. Ramsay, A.J., Leong, K.H., Boyle, D., Ruby, J., Ramshaw, I.A., 1994. Enhancement of mucosal IgA responses by interleukin-5 and interleukin-6 encoded in recombinant vaccine vectors. Reprod. Fertil. Dev. 6, 389 – 392. Rowland-Jones, S.L., Nixon, D.F., Aldhous, M.C., Gotch, F., Ariyoshi, K., Hallam, N., Kroll, J.S., Froebel, K., McMichael, A., 1993. HIV-specific T-cell activity in an HIV-exposed but uninfected infant. Lancet 341, 860 – 861. Rowland-Jones, S.L., Sutton, J., Ariyoshi, K., Dong, T., Gotch, F., Mcadam, S., Whitby, D., Sabally, S., Gallimore, A., Corrah, T., Takiguchi, M., Schultz, T., McMichael, A., Whittle, H., 1995. HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women. Nat. Med. 1, 59 – 64. Schneider, T., Ullrich, K., Bergs, C., Schmidt, W., Riecken, E.O., Zeitz, M., 1994. Abnormalities in subset distribution, activation and differentiation of T-cells isolated from laarge intestine biopsies in HIV infection. Clin. Exp. Immunol. 95, 430 – 435. Smith, P.D., 1994. Role of the mucosa in human immunodeficiency virus disease. Mucosal Immunol. Update 2, 3 – 6. Sturgess, C.P., 1997. Studies on mucosal effector mechanisms in feline immunodeficiency virus (FIV) infection in cats. PhD thesis, University of Bristol. Sutjipto, S., Pedersen, N.C., Miller, C.J., Gardner, M.B., Hanson, C.V., Gettie, A., Jennings, M., Higgins, J., Marx, P.A., 1990. Inactivated simian immunodeficiency virus-vaccine failed to protect rhesus macaques from intravenous or genital mucosal infection but delayed disease in intravenously exposed animals. J. Virol. 64, 2290 – 2297. Targan, S.R., Deem, R.L., Liu, M., Wang, S., Nel, A., 1995. Definition of a lamina propria T-cell responsive state: enhanced cytokine responsiveness of T-cells stimulated through CD2 pathway. J. Immunol. 154, 664 – 675. Ulmer, J.B., Donnelly, J.J., Parker, S.E., Rhodes, G.H., Felgner, P.L., Dwarki, V.J., Gromkowski, S.H., Deck, R.R., Dewitt, C.M., Friedman, A, Hawe, L.A., Leander, K.R., Martinez, D., Perry, H.C., Shiver, J.W., Montgomery, D.L., Liu, M.A., 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259, 1745 – 1749. Walker, C.M., Moody, D.J., Stites, D.P., Levy, J.A., 1986. Lymphocytes-CD8 + can control HIV infection in vitro by suppressing virus replication. Science 234, 1563 – 1566. Willett, B.J., Picard, L., Hosie, M.J., Turner, J.D., Adema, K., Clapham, P.R., 1997a. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. J. Virol. 71, 6407 – 6415. Willett, B.J., Hosie, M.J., Neil, J.C., Turner, J.D., Hoxie,
C.R. Stokes et al. / Journal of Biotechnology 73 (1999) 213–221 J.A., 1997b. Common mechanisms of infection by lentiviruses. Nature 385, 587. Willett, B.J., Flynn, J.N., Hosie, M.J., 1997c. FIV infection of the domestic cat: an animal model for AIDS. Immunol. Today 18, 182 – 188.
.
221
Wilson, A.D., Bailey, M., Williams, N.A., Stokes, C.R., 1991. The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocvanin using cholera toxin as an adjuvant. Eur. J. Immunol. 21, 2333 – 2339.
.